Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas

Abstract

Background: Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.

Methods: The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.

Results: 39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.

Conclusions: Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856.

Figure 1

Ig mRNA levels increase with B cell differentiation. As B lymphocytes pass through stages of maturation from precursor B cells to naïve B cells to germinal center cells to post-germinal center cells then to plasma cells, the level of mRNA encoding immunoglobulin increases. Current slide-based methods are generally able to detect the mRNA levels found in the later stages of differentiation (generally in the post-germinal center stages).

Figure 2

2-Color RNA Detection Methodology. A cocktail of antisense hapten-labeled riboprobes, targeting the constant region of either KAPPA or LAMBDA mRNA, was hybridized to de-paraffinized protease treated tissue samples. Following stringency washes the haptens were sequentially detected using a cognate anti-hapten monoclonal antibody conjugated to HRP which catalyzed deposition of a tyramide-hapten conjugate. Following inactivation of the HRP conjugate, each amplified hapten was then sequentially detected using a second anti-hapten antibody-HRP conjugate which catalyzed silver or pink chromogen deposition.

Figure 3

Staining Pattern in Reactive Lymphoid Tissues. A: Low power view of tonsil showing polyclonal staining within germinal center and mantle zone, and scattered paracortical B cells as well as intra-germinal center and paracortical plasma cells (40×). B: High power view of germinal center demonstrating polyclonal centrocytes, centroblasts, and a few plasma cells (500×). C: High power view of medullary sinuses with polyclonal plasma cells (600×). D: High power view of reactive paracortical hyperplasia showing KAPPA restricted B-immunoblast (black arrow), LAMBDA restricted B-immunoblast (pink arrow), negatively staining presumed T-immunoblast (blue arrow), and negatively staining histiocyte (blue arrowhead) (1000×).

Figure 4

Light chain restriction in B cell lymphomas. A-B: CLL/SLL (A) residual germinal center on left with sheets of monoclonal KAPPA light chain restricted lymphoma cells on the right (100×); (B) higher power view of KAPPA light chain restricted cells (500×). C-E: DLBCL (C) diffuse sheets of LAMBDA light chain restricted cells (100×); (D) same case showing intermediate magnification of LAMBDA light chain restricted lymphoma cells (400×); (E) same case showing higher power view with centroblastic morphology and LAMBDA light chain restriction. Rather than an inherent difference in the chromogens, but rather due to the larger cell size and lower nuclear to cytoplasmic ratios in DLBCL as compared to SLL or FL, the cellular detail is easier to appreciate in E than in images B or H (600×). F-H: Follicular lymphoma (F) low power view showing neoplastic germinal centers with KAPPA light chain restriction and cuff of polyclonal plasma cells (100×); (G) same case at intermediate power showing KAPPA light chain restricted FL; (H) same case at higher power showing KAPPA light chain restriction of both small and large neoplastic cells (600×).

Figure 5

Non-specific nucleolar staining with lambda probe. A-C: Germinal center in a reactive tonsil. (A) polyclonal mixture of KAPPA and LAMBDA positive cells (400×); (B&C): arrows point out KAPPA expressing cells as evidenced by black ring of cytoplasmic staining with non-specific pink nucleolar staining (600×). D-E: Follicular lymphoma. (D) low power view of FL case which was indeterminate for KAPPA and lambda due to expressing neither light chain (40×); (E): high power view of same case demonstrating lack of cytoplasmic staining pattern for either KAPPA or LAMBDA light chain expression with non-specific pink nucleolar staining (600×).